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Colony PCR
A faster, easier alternative to Smash N Grab Yeast Genomic DNA Prep followed by PCR. HOW DOES PCR WORK? Please, please, please know this if you are using this technique. Start HERE http://www.dnalc.org/resources/animations/pcr.html Overview This is a quick and easy yeast colony PCR protocol that does not require zymolyase step. This new version of the protocol uses 10uL PCR reactions, significantly reducing the reagent costs. Older version: Blackburn Lab: Quick and Easy Yeast Colony PCR We (Rick, Brett, and Mark) have done this 150 lanes and counting now and if anything it works more consistently than Smash and Grab. Materials *Standard PCR Machines, tubes *Qiagen Taq Polymerase Kit with Q-solution *This Qiagen Kit is stocked in Biochemistry Stores at UW. *dNTPs 10mM ea. *Home-made Taq and buffer have been used successfully with this protocol, but the addition of the Q-solution, which is mainly betaine, is criticial. *A small yeast colony *0.02M NaOH (10uL per reaction) *Multi-channel pipettes are very helpful. test *Colony PCR Calculator Procedure Yeast Cell Lysis #Aliquot 10uL (per well) of 0.02M NaOH into PCR tubes (our stock is usually 1M NaOH so you will need to do a dilution). #Using a sterile pipette tip, pick a small colony and resuspend in NaOH. #*If the solution is cloudy, you've added enough cells. #*I have been told adding too much yeast can inhibit the reaction. In our optimization in the Kennedy Lab, this was never the case- a wide range of starting amounts all worked great. They may have been referring to the problem of transferring yeast accidentally with the supernatant, noted below in PCR.3. #Boil the samples on a PCR machine by incubating the tubes at 99C for 10 minutes. #*'Mark your boil PCR strips with sharpie over the entire top of the strip, and your PCR reaction strips only on one side or in some way that is distinguishable from the boiled strips so they don't get mixed up.' #*After removing your boiled strips you will want to spin down the strips for about 1-2 seconds. You can use the adapter and centrifuge on Monique's bench but PLEASE balance your samples. #*In the mean time, prepare the master mix for the PCR reaction. #*The boiled samples are stable at room temp for some time. Keep on ice or freeze for longer. PCR Qiagen's PDF handbook for this kit is here, PDF genomic DNA guidelines for this kit is here. #Prepare the master mix solution containing (tip: add the largest volumes first): #*2uL 5X Q-solution #*1uL 10X PCR Buffer - this is the clear 10X buffer, NOT the red CoralLoad 10X also in the kit. #*0.2uL dNTPs (10mM each) #*0.2uL foward primer (100uM) #*0.2uL reverse primer (100uM) #*0.1uL Taq #*5.3uL ddH2O Note: There is a chart above Bhumil and Mark's benchtop that allows you to make larger reaction sizes (x10, x20 etc.) If you are doing a large PCR with multiple primers, you may want to make a large master mix (excluding the primers) and then separate into smaller reaction mixes and add the primers to their respective tubes. MAKE SURE YOU FLICK THE TUBES BEFORE ALIQUOTING TO PCR TUBES OR THEY WILL NOT BE MIXED AND YOUR PCR WILL NOT WORK. #Aliquot 9uL of the master mix solution into fresh PCR tubes. #Transfer 1uL of boiled samples to the master mix aliquots (a multi-channel pipette is helpful here). Pull from the supernatant of your boiled samples, being careful not to pipette any of the yeast from the bottom. #Run the following PCR cycle: ##3 min at 94C ##30 cycles of: ###1 min sec at 94C ###1 min at appropriate annealing temperature ###1 min/kbp at 72C (I generally do 2 minutes) ##10 min at 72C ##stay at 10C indefinitely NOTE: If you are doing a PCR of a strain that is deleted for KanMX the approximate bp of the marker is 1600, so the minimum time should be 2 minutes! Notes *Q-solution is critical for this protocol; the main ingredient is betaine. *Note that the primer concentration we use is about ten-times more than standard PCR protocols. *The amount of taq used in this updated protocol is more per volume than the original protocol, but still less overall. *For restriction digestion of the PCR products, use 2uL of the PCR reaction in 20uL total volume. *The expected PCR product size should be as short as possible. Anything less than 1kbp can be easily amplified. *Generally, 2 distinct PCR products can be amplified in a single reaction. I do this to check the 5' and 3' ends of an integration in a single reaction (4 primers and different expected product sizes). This fails in about 5% of primer sets. N.B.: The Eppendorf PCR machines will not turn off when you open them and take your samples out! You have to find the cycler you were using on the menu screen (i.e., Cycler 1 or Cycler 2) and make sure you stop the program. Specifically, many programs have a 4C or 10C hold step at the end, and the machine will stay in that step unless you abort the program to end it, after you take your samples out.